Group 3Fabiha Aziz

Fariha Munir

Fatima Tuz Zahra

Fatima Tuz Zahra Khan

Muhammad Ahmad

Saneea Imran

Tatheer Fatima

Contents

1.Introduction of the disease or disorder2.Types of mutations involved3.Conventional treatment4.Type of vector5.The procedure of gene therapy6.History of clinical trials7.Commercially available treatments

Introduction

•What is hemophilia?•What is does to our body?•What causes hemophilia?

What is hemophilia?

• Hemophilia is an inherited bleeding disorder in which the blood fails to clot effectively.• Hemophiliacs do not bleed more extensively or more quickly than the normal people but they do bleed for a longer time after an injury or surgery.

What it does to our body?

• The bleeding may be internal or external• Internal bleeding occurs in knees ankles tissues and muscles. If left untreated the bleeding in the joints may lead to agonizing pain• And can be fatal when the bleeding occurs in the vital organ like brain

Types of Hemophilia

Hemophilia A is a recessive X-linked genetic disorder

involving a lack of functional clotting

Factor VIII and represents 80% of hemophilia cases.

Hemophilia B is a recessive X-linked genetic disorder

involving a lack of functional clotting

Factor IX. It comprises

approximately 20% of

hemophilia cases.

Hemophilia C is an autosomal

genetic disorder involving lack of

functional Clotting Factor XI.

Hemophilia C is not completely recessive, as

heterozygous individuals also show

increased bleeding.

Hemophilia A

• All races• Affects men and women become carriers as gene is present on the X chromosome.

Moderate cases minor traumas are usually the

cause of the cause of bleeding

Milder have fewer

hemorrhages

Factor Replacement Therapy

Infusion of factor 8-9 to prevent or control bleeding

Plasma derived factors and genetically engineered

MUTATIONBy: Fatima Tuz Zahra

Mutation of Hemophilia A

• Coagulation factors are proteins that work together in the blood clotting process. • F8 gene provides instructions for making a coagulation factor VIII•Mutations in the F8 gene lead to the production of an abnormal version of coagulation factor VIII.

28 region of X Chromosome.

Inversion and Crossing Over of Intron22

Conventional Treatments

By: Tatheer Fatima

Conventional treatment

The two main approaches to treatment are• Preventive treatment : Refers to the regular medications that are taken to prevent episodes of bleeding and subsequent complications

• Episodic treatment : Used to manage episodes of prolonged bleeding.

Conventional treatment

• Treatment With Replacement Therapy :Concentrates of clotting factor VIII (hemophilia A) or clotting factor IX (hemophilia B) are slowly dripped or injected into a vein

• Preparation of clotting factor concentrate :Human Blood Clotting factor concentrate Recombinant Clotting factors

Conventional treatment

Types of Replacement therapy :

• Preventive or prophylactic therapy:Replacement therapy on a regular basis to prevent

bleeding

• Demand therapy:only need replacement therapy to stop bleeding when it occurs

Conventional treatmentPROS

1. Quicker treatment. Early treatment lowers the risk of complications

2. Fewer visits to the doctor or emergency room

3. Costs less than treatment in a medical care setting

4. Helps children accept treatment and take responsibility for their own health

CONS1. Antibody(proteins)

that attack the clotting factor

2. Viral infections from human clotting factors

3. Damage to joints, muscles, or other parts of the body resulting from delays in treatment

Conventional treatment

Desmopressin•Man made hormone• Can treat mild hemophilia A • Cannot treat hemophilia B or severe hemophilia A• Stimulates the release of stored factor VIII• Injection or as nasal spray• Only in certain situations. e.g. you may take this medicine prior to dental work or before playing certain sports to prevent or reduce bleeding

Conventional treatment

Anti fibrinolytic Medicines• Tranexamic acid and epsilon aminocaproic acid• Can be used with replacement therapy• Given as a pill and they help keep blood clots from breaking down.•Most often are used before dental work or to treat bleeding from the mouth or nose or mild intestinal bleeding.

Types of vectorBy: Muhammad Ahmed

Ex-vivo

• Cells transduced in culture, then returned to patient• Greater control over transfection conditions• Unchanged cells can be screened out• Difficult to transpant

In-vivo

• Cells modified within the body• Introduction of vector with genetic material in patient’s body• No transplantation issues• Cost-effective•May provoke immune system• Ultimate goal for gene therapy

Viral Vectors

Retroviral vectors•Moloney murine leukemia virus (MoMLV)-based• Can infect wide variety, integrate into host genome, relatively non-immunogenic• Only in actively dividing cells• Problems: insertional mutagenesis, inactivation by complement

Adenoviral vectors

• Relatively large, double-stranded DNA viruses• Infect non-dividing cells• Can transfer multiple copies of gene, in vivo• Problems: Many humans may be immune to virus, not integrated into host genome

Adeno-associated (AAV) vectors

• Relatively small, single stranded DNA parvovirus• Viral coding sequence replaced by transgene• Non-dividing cells

Lentiviral Vectors

• Integrate into genome, infect non-dividing cells• Problems: limited range of cell targets, pathogens!• HIV – big risk of recombination infectious virus

Non-Viral Vectors

• Through plasmids• Problem: Transient expression of genes (several days)

• Direct injection of naked DNA• Problems: Low efficiency of transduction, not integrated into genome

Procedure of Gene Therapy for Hemophilia A

By: Saneea Imran

Gene therapy for hemophilia A

• Hemophilia is an interesting target for gene therapy as the genetics of the disease are well understood.

• Introducing a functional copy of the mutated gene through a vector can provide an effective cure.

Clotting factor level = Clinical Phenotype

Gene therapy for Hemophilia A

• Vector: Both viral and non-viral vectors can be used

• Target Cell: Hematopoietic stem cells, hepatocytes, skeletal muscle cells, endothelial cells.

• Approach: Both in-vivo and ex-vivo gene therapy approach can be used.

Non-Viral vector for gene therapy of Hemophilia A• Year: 2001• Non-viral somatic cell gene therapy for hemophilia A• Vector used: Plasmid• Cells targeted: Dermal fibroblasts

Procedure

Restriction enzymes

Preparing the vector

Desired gene that produces Factor VIII

Gene of

interest

Antibiotic gene Patient with

HemophiliaDermal fibroblasts

of patient ( skin biopsy )

TransfectionCells that

successfully produce FVIII are selected

and inserted

into patient

Viral Vectors for gene therapy of Hemophilia AVectors that have been used are:1. Retroviral and Lentiviral Vectors2. Adeno Associated Viral Vectors

Using Retroviral Vectors

Producing recombinant Retroviral vectors

Targeting cells with Retrovirus

Clinical TrialsBy: Fatima Tuz Zahra Khan

Stem cell-based gene transfer

• Implantation of Genetically Modified Stem Cells• Genetic modification and transplantation of hemopoietic stem cells (HSCs)• Use of Retroviral and lentiviral vectors• Trials remain un-successful due to insufficient levels of FVIII

In Vivo methodsRetroviral vectors• Retroviral vectors transduce replicating cells by stably

integrating into the host genome.• Preclinical studies involving rabbits and dogs did show

possible benefits.Routes?• Subjects received a peripheral intravenous infusion of

retroviral vector carrying a B domain–deleted human FVIII gene.

Efficiency?• 9 of 12 subjects showed a FVIII level above 1%• Insertional mutagenesis and the risk of subsequent

malignancy.• Lack of efficacy in one clinical phase 1 trial in humans.

LENTIVIRAL VECTORS

• Lentiviruses efficiently transduce nondividing cells.• Overcoming a hurdle faced by retroviral vector systems.• Routes?• In murine models, intraperitoneal administration of lentivirus transduced liver, spleen, blood and bone marrow• Efficieny?• four- to sixfold rise in expression• Concerns over genomic integration and subsequent malignancy.

ADENOVIRAL VECTORS

• One of the most efficient vectors• Transducing dividing and nondividing cells, with a tropism

for hepatocytes• Genetic payload capacity making them ideal vectors for

the FVIII geneRoutes?• Intramuscular Injection.• Liver-directed administrationEfficiency?• Single Human Trail• Suspended due to unacceptable toxicity not predicted by

mice models

Adeno associated Virus:

• Success in mouse and other large animal models.• Limitations due to small genome size.Routes?• Intramuscular Injection.• Liver-directed administrationEfficiency?• Two trials have examined the use of AAV-2 vectors in

human subjects• Limited efficacy

Commercially available Treatments

Treatment

• Regular infusions of DDAVP or clotting factor• Clot-preserving medications (antifibrinolytics)• Fibrin sealants• Physical therapy• First aid for minor cuts• Vaccinations

Clinical Trials

• Currently are for hemophilia B• AAV vector encoding factor IX (FIX) under the control of a liver-restricted promoter• Promising results

Problems

• First clinical trial of hepatic artery infusion of an AAV2-FIX vector quickly encountered obstacles that had not been identified in the preclinical studies• Risk of vertical transmission• Risk for potential offspring if the spermatocytes are transduced

References

• Haemophilia A: from mutation analysis to new therapies• Jochen Graw, Hans-Hermann Brackmann, Johannes Oldenburg, Reinhard

Schneppenheim, Michael Spannagl & Rainer Schwaab• Genetics Home Reference • (http://ghr.nlm.nih.gov/condition/hemophilia)

• http://www.visibleproductions.com/index.php?page=asset_detail&asset_id=vpl_0715_001